All-trans-beta-retinoic acid (RA) has both comitogenic and antiproliferative effects on murine Swiss 3T3 cells. Treatment of quiescent 3T3 cells for less than 24 hr with micromolar concentrations of RA potentiates subsequent mitogenic response of those cells to phorbol 12-myristate 13-acetate. Longer exposures of 3T3 cells to RA result in inhibition of DNA replication as measured by [3H]thymidine incorporation and decreased growth rates and saturation densities for cells grown in either 2% or 10% serum. Both the comitogenic and antiproliferative activities of RA for 3T3 cells are RA-dose dependent. RA-induced decreases in the 3T3 cell saturation density are reversible only after resuspension of cells by trypsinization and replating. Treatment of 3T3 cells for 48 hr with RA inhibits the rate of [3H]thymidine incorporation by 35--50%, while autoradiographic data show that labeling indices are similar to control values. Equal percentages of control and 48-hr RA-treated quiescent 3T3 cells respond to subsequent stimulation with 10% serum as determined by autoradiographic and flow cytometric analyses. However, the progression of RA-treated cells through the S phase of the cell cycle is slowed. These data suggest that inhibition of 3T3 cell proliferation by RA is established after a minimum 24-hr treatment and that this inhibition is the result of a decreased rate of DNA replication in S-phase cells.